1. Field of the Invention
The present invention relates to a reagent composition used for assaying hydrogen peroxide contained in a liquid, particularly, a body fluid.
2. Description of the Prior Art
Currently, hydrogen peroxide assay is very extensively used in clinical examinations in the medical field and in various other fields such as the chemical industry, food industry and public sanitation industry. Particularly in the clinical examinations are, the methods for analyzing body fluids with enzymes have spread rapidly. Among these methods, the hydrogen peroxide assay is very important, since numerous components of the body fluid can be assayed by a relatively simple method wherein the body fluid is reacted with an oxidase to form hydrogen peroxide and the resulting hydrogen peroxide is measured. Typical combinations of a component/oxidase include, for example, glucose/glucose oxidase, cholesterol/cholesterol oxidase, uric acid/uricase, amino acid/amino acid oxidase and pyruvic acid/pyruvic acid oxidase.
The hydrogen peroxide thus formed may be assayed by various methods. The most generally employed method comprises oxidizing a chromogen in the presence of a peroxidase to form a pigment and determining the latter colorimetrically. The chromogen thus selected generally has amino and hydroxyl groups in the molecule, a high stability and a high pigment-forming power. The most frequently used chromogen is a combination of 4-aminoantipyrine and phenol known as "Trinder reagent". Recently, numerous modifications of this chromogen have been reported.
In the field of so-called "dry chemistry" in which a reagent composition used for the assay of a body fluid composition is fixed on a carrier, a benzidine derivative, particularly o-tolidine has been preferably used as the chromogen. This is because the use of this substance as the chromogen is advantageous in that a quite sharp sensitivity is obtained and a pigment which has a maximum absorption on the long wave-length side in the visible light region is formed with the result that it is not easily influenced by various coloring components present therein. However, o-tolidine is thought to be carcinogenic so that a special care must be taken in the production of testing implements containing this compound.
Recently, a chromogen having the characteristic properties of o-tolidine but free of carcinogenicity has been developed. This is a benzidine compound having methyl groups in positions 3, 3', 5 and 5' [see V. R. Holland et al., "Tetrahedron", 30, 3299 to 3302 (1974)]. Further, it is disclosed in the specification of West German Pat. No. 2,460,903 that benzidine substituted with alkyl groups other than methyl group in positions 3, 3', 5 and 5' is also usable as the chromogen.
However, these benzidine derivatives have a quite poor solubility in water and, therefore, they can be used in only a limited concentration range in an acidic pH region. This is a serious defect, since the enzymatic assay of body fluids is effected in a neutral region in most cases. It is because of this defect that the benzidine derivatives have been used mainly for the preparation of test pieces only. However, even the test pieces containing the benzidine derivatives cannot completely overcome the disadvantages due to the above-mentioned defect so that they are usable only when the amount of hydrogen peroxide formed is very small.
Attempts have been made for broadening the concentration range of hydrogen peroxide to be assayed, one of which is a process wherein the benzidine derivatives are used in admixture with another chromogen (see the specification of Japanese Patent Publication No. 5520/1982).
The poor solubility of 3,3',5,5'-tetraalkylbenzidine in water causes another disadvantage in the preparation of the test pieces. For example, when 3,3',5,5'-tetraalkylbenzidine is selected as the chromogen in the preparation of an aqueous impregnation solution generally comprising an enzyme, a buffer and a chromogen in the course of the preparation of the test pieces for assaying glucose in a body fluid, it is quite difficult to obtain a homogeneous, aqueous impregnation solution. Processes which have been proposed to overcome this defect include one which comprises two steps of impregnation of the enzyme and the buffer and impregnation of the chromogen (see the specification of Japanese Patent Publication No. 29160/1982) and one wherein a mordant is added to the aqueous chromogen impregnation solution (see the specification of U.S. Pat. No. 4,361,648).
After intensive investigations made with due regard to the above-mentioned defects, the inventors have completed the present invention.